Tamoxifen and retinoid regulation of antiproliferative pathways in high risk human breast epithelium cells (HRBEC) and in breast cancer cells is being studied to characterize high risk cells and to develop new treatment strategies for the chemoprevention and treatment of breast cancer. Studies in progress have demonstrated that HRBEC are growth inhibited by all-trans retinoic acid (AT), and that this inhibition is due to blockade of cell cycle progression at G0G1 without stimulation of apoptosis. AT modulation of protein expression of the retinoic acid receptors RAR-alpha, RAR-beta, RAR-gamma, of transforming growth factor beta (TGF-beta), and cyclin D1, cdk4, and retinoblastoma proteins have been studied to define the mechanism of action of these retinoids. The findings indicate the antiproliferative effects on expression of retinoic acid receptors and cell cycle regulatory proteins in normal high risk breast epithelial cells are intermediate between normal (nonrisk) breast epithelial cells and breast cancer cells, and thus may represent early changes in carcinogenesis. To further define the characteristics of high risk mammary epithelium, an intramural clinical trial is in progress to establish short-term mammary epithelial cell lines from women at high risk for breast cancer (including BRCA-1 positive). The growth, metabolic, and molecular properties of these cells will be examined. The effects of tamoxifen and retinoic acid on cell cycle progression, cell differentiation, and apoptosis of human breast cancer cells is also being studied to define the synergistic effect of these agents and to identify new targets for the treatment of breast cancer. Studies in progress have demonstrated that AT acts synergistically with tamoxifen to regulate multiple antiproliferative pathways in breast cancer cells, including a time-dependent early (day 4) stimulation of TGF-beta secretion and induction of cell differentiation, followed by a late (day 7) downregulation of the apoptotic protein bcl-2 and stimulation of apoptosis. Cell cycle progression was not synergistically regulated, indicating selective recruitment of the antiproliferative pathways. TGF-beta is an important mediator of TAM action on breast cancer cells, which suggested a potential role in the synergistic effects of AT/TAM in combination. This was studied and revealed that TGF-beta mediates the synergistic antiproliferative effects of TAM and AT in combination, and by a novel autocrine action of TGF-beta: combining with AT to downregulate bcl-2 protein expression and stimulate apoptosis. Studies are in progress to define the mechanism of AT/TGF-beta interaction. These findings also suggest several important targets (cell differentiation antigens, TGF-beta, bcl-2) which may be utilized for new treatment strategies for breast cancer. The estrogen receptor and retinoic acid receptor genes are methylated in their 5 regions and not expressed in hormone-independent human breast cancer, rendering them unresponsive to estrogen, antiestrogens, and retinoids. We are studying the ability of demethylation of these genes to induce hormone responsiveness, with the aim of further defining the steroid receptor pathways and their interactions in these cells, and the identification of new treatment strategies for hormone-independent breast cancer. Studies to date indicate that treatment of estrogen receptor negative breast cancer cells (MDA-MB-231) with 5-aza-2-deoxycytidine (deoxyC) rapidly demethylates the ER and RAR genes (within 72 hours, as determined by methylation-specific PCR), and induces both mRNA and protein expression of the respective genes. Gene expression is enhanced by cotreatment with deoxyC and the histone deacetylase inhibitor tricostatin (TSA). Studies on cell growth indicate that deoxyC, in the presence of fetal calf serum, is growth inhibitory, and this inhibition is enhanced by the addition of TSA. Studies are in progress to determine the role of induced receptor protein (ER and RAR) in mediating the responsiveness of demethylated cells to estradiol, antiestrogens and retinoids, including the selectivity of retinoid action, contribution of specific RAR isoforms to the cellular response, the mechanism of cellular inhibition, synergistic action of retinoids and antiestrogens in combination, and cross-modulation between the estrogen and retinoic acid receptor pathways.